Biodegradable microsphere-hydrogel ocular drug delivery system

ABSTRACT

A hydrogel delivery composition, including degradable microcapsules suspended in a degradable thermo-responsive hydrogel. The hydrogel is thermo-responsive at a physiological temperature and changes after application to a more solid state due to body temperatures. The composition includes one or more treatment agents to be released over time as the composition degrades. The composition can be varied to modify the structure and/or release of the treatment agent.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication, Ser. No. 62/232,545, filed on 25 Sep. 2015. The ProvisionalPatent Application is hereby incorporated by reference herein in itsentirety and is made a part hereof, including but not limited to thoseportions which specifically appear hereinafter.

FIELD OF THE INVENTION

This invention relates generally to drug or other treatment deliveryand, more particularly, to a hydrogel delivery composition and methodthat can deliver treatment materials in a controlled matter for anextend period of time.

BACKGROUND OF THE INVENTION

Choroidal neovascularization (CNV) secondary to age-related maculardegeneration (AMD) is a leading cause of vision loss in elderly patientsin developed nations. It has been demonstrated that vascular endothelialgrowth factor (VEGF) plays a key role in the pathophysiology of thedisease. The U.S. Food and Drug Administration has approved severalanti-VEGF therapeutics to treat CNV secondary to AMD, namely ranibizumab(Lucentis, Genentech, South San Francisco, Calif., USA) and aflibercept(Eylea®, Regeneron, Tarrytown, N.Y., USA). Additionally, the use ofanti-VEGF therapeutics has increased for other indications such asdiabetic macular edema (DME) and vein occlusion. Due to these successfuloutcomes, it is not surprising that there has been a paradigm shift inthe use of pharmacological therapeutics over surgical techniques duringthe last decade to treat visually devastating diseases.

The current standard to deliver anti-VEGF therapeutics is through amonthly (or bimonthly) bolus intravitreal injection. With eachinjection, serious potential complications can arise includingendophthalmitis, retinal detachment, intravitreal hemorrhage, andcataract. To improve upon the socio-economic impact associated withrepeated injections and to further lower the risk of associatedpotential complications, there is a need to reduce the number andfrequency of injections.

A multitude of drug delivery systems (DDSs) have been successful incontrolling and extending the release of various model drugs. A commontheme among these DDSs is the use of polymeric microspheres to controlrelease. However, when microspheres are injected into the eye asindependent units, they can become lodged in ocular tissues (e.g.,trabecular meshwork), which may cause unintended and seriouscomplications. Thus, there is a continuing need for improved drugdelivery systems, particularly in the development of intraocular DDSs.

SUMMARY OF THE INVENTION

A general object of the invention is to provide an improved deliverysystem and method for delivering treatment material(s), such as on orwithin the eye.

The general object of the invention can be attained, at least in part,through a delivery composition including degradable microcapsules (e.g.,nano- or micro-spheres or other similar microencapsulation structures)suspended in a degradable thermo-responsive hydrogel. The hydrogel isthermo-responsive in that it desirably changes its physical state from aliquid-like state at room temperature to a more solid state at bodytemperature (e.g., at a physiological temperature of about 32° C. toabout 37° C.). The microcapsules encapsulate and release a treatmentagent.

The rate of release can be controlled via the rate of degradation, whichcan be controlled, for example, by altering a chemical component ratio,the molecular weight of the polymer(s), surface modification, and/ormanufacturing procedures. In some embodiments a non-encapsulatedtreatment agent can optionally be additionally dispersed within thehydrogel, and/or microcapsules having at least two different releaserates can be used together.

Embodiments of this invention focus on anti-VEGF as a treatment agent,but the invention can also be used to deliver other drugs, such asophthalmic drugs (for example, antibiotics for ocular surgery),anti-platelet-derived growth factor (anti-PDGF) agents, cells, deliverycells, a corticosteroid, enzymes, peptides, nucleic acids, orcombinations thereof. The invention can, for example, reduce oreliminate patient administration of treatment after ocular surgery, suchas eliminating the need to administer topical (eyedrop) antibiotics forseveral days. Most often, the patient compliance is low and at risk forinfection.

The invention further comprehends a method of delivering a compound toan eye, including: applying to or into an eye of a mammal a compositionin a first physicochemical state, wherein the composition comprises amicroencapsulated treatment agent suspended in a thermo-responsivehydrogel; and the composition changing to a second physicochemical stateupon administration, wherein the second physicochemical state is moresolid than the first physicochemical state and degradable to release themicroencapsulated treatment agent. The applied microcapsules release thebioactive treatment agent over time after application.

The method can be used with any composition described herein. Inembodiments of this invention, the degradation can be controlled by thetype of and/or amount of crosslinking within the composition, therebycontrolling the release the microencapsulated treatment agent. Usingmore than one microencapsulated treatment agent, such as differentmicroencapsulations for a same agent type, and/or also placingnon-encapsulated agent in the hydrogel itself, can also or further beused to provide a desired release.

Compositions of this invention can be applied by any suitable manner,desirably in a first physicochemical state, such as by intravitrealinjection, by periocular or transcleral injection, by topicalapplication including scleral structures, by intracameral application,by suprachoroidal application, within ocular implants, or combinationsthereof. Injections can be made by small gauge needle (e.g., 20 gauge to33 gauge) or by microcatheter.

Other objects and advantages will be apparent to those skilled in theart from the following detailed description and examples taken inconjunction with the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows: A) a representative SEM image of ranibizumab-loadedmicrospheres (scale bar: 2 μm); B) a ranibizumab-loaded DDS in a 28Gneedle; C) a representative SEM image of aflibercept-loaded microspheres(scale bar: 2 μm); and D) an aflibercept-loaded DDS in a 28G needle atroom temperature.

FIG. 2 includes graphs of: A) cumulative percent-release of ranibizumabfrom microspheres (“alone”) and microspheres suspended in hydrogel (“inhydrogel”), where the microspheres in hydrogel had a significantly lowerIB and a steadier, extended release; and B) cumulative drug-release frommicrospheres in hydrogel, where after Day 7, 0.153 μg/day of ranibizumabwas released and an additional, 25.1±12.1% (27.3 μg) of encapsulateddrug remained entrapped in the hydrogel after complete degradation ofthe microspheres (circled). Error bars represent standard error (n=3).

FIG. 3 includes graphs of: A) cumulative percent-release of afliberceptfrom microspheres (“alone”) and the effect of suspending microspheres inhydrogel (“in hydrogel”), where flibercept-loaded microspheres suspendedin hydrogel had a significantly lower IB and a steadier, extendedrelease; and B) cumulative drug-release from microspheres in hydrogel,where after the first week, 0.065 μg/day of aflibercept was released,and an additional, 32.7±1.1% (21.4 μg) of encapsulated drug remainedentrapped in the hydrogel after complete degradation of the microspheres(circled in red). Error bars represent standard error (n=3).

FIG. 4 shows bioactivity of the ranibizumab-loaded DDS (“Rani.-gel”).Significant bioactivity (p<0.05) is seen throughout release ofranibizumab compared to the drug-free DDS (“Blank-gel”; right). Errorbars represent standard error (n=16, left; n=6, right).

FIG. 5 shows bioactivity of the aflibercept-loaded DDS (“Aflib.-gel”).Significant bioactivity (p<0.05) is seen throughout release ofaflibercept compared to the drug-free DDS (“Blank-gel”; right). Errorbars represent standard error (n=16, left; n=6, right).

FIG. 6 shows release of ranibizumab from biodegradable microspheres andbiodegradable thermo-responsive hydrogel, where different amounts ofcrosslinker were used to change the degradation rate.

FIG. 7 shows an example of a CNV lesion area quantification technique:A) the original image opened in ImageJ and three levels are selected.AfterMOT, three images are produced: B) Region 0 (background), C) Region1 (diffuse leakage), and D) Region 2 (CNV). With Region 2 selected, thethreshold is adjusted to include all pixels within the image. The lesionis outlined (square in E) and the number of pixels within the lesion ismeasured, which is then converted into an area.

FIG. 8 shows average single flash ERG intensity-response functions fornon-treated animals (top, a-wave; bottom, b-wave). Lines are the resultsof the Naka-Rushton analysis to the cumulative pool of data points andsymbols represent different time points of investigated time frame. Nosignificant differences were seen in either maximal a- and b-waveresponse or half-maximal a- and b-wave response.

FIG. 9 shows average single flash ERG intensity-response functions fordrug-free DDS treated animals (top, a-wave; bottom, b-wave). Lines arethe results of the Naka-Rushton analysis to the cumulative pool of datapoints and symbols represent different time points of investigated timeframe. A significant difference in maximal b-wave response is seenbetween Control and Week 12 (p<0.05). No significant difference inhalf-maximal a- or b-wave response was observed.

FIG. 10 shows average single flash ERG intensity-response functions forranibizumab-loaded DDS treated animals (top, a-wave; bottom, b-wave).Lines are the results of the Naka-Rushton analysis to the cumulativepool of data points and symbols represent different time points ofinvestigated time frame. No significant differences were seen in eithermaximal a- and b-wave response or half-maximal a- and b-wave response.

FIG. 11 shows average single flash ERG intensity-response functions forbolus ranibizumab treated animals (top, a-wave; bottom, b-wave). Linesare the results of the Naka-Rushton analysis to the cumulative pool ofdata points and symbols represent different time points of investigatedtime frame. No significant differences were seen in either maximal a-and b-wave response or half-maximal a- and b-wave response.

FIG. 12 shows average single flash ERG intensity-response functions foraflibercept-loaded DDS treated animals (top, a-wave; bottom, b-wave).Lines are the results of the Naka-Rushton analysis to the cumulativepool of data points and symbols represent different time points ofinvestigated time frame. A significant difference in maximal a-waveresponse is seen between Control and Week 1-Week 12 (p<0.05). Nosignificant difference in half-maximal a- or b-wave response wasobserved.

FIG. 13 shows average single flash ERG intensity-response functions forbolus aflibercept treated animals (top, a-wave; bottom, b-wave). Linesare the results of the Naka-Rushton analysis to the cumulative pool ofdata points and symbols represent different time points of investigatedtime frame. A significant difference in maximal a-wave response is seenbetween Control and Week 1-Week 12 (p<0.05). Additionally, a significantdifference in maximal b-wave response is seen between Control and Week1-Week 12 (p<0.05). No significant difference in half-maximal a- orb-wave response was observed.

FIG. 14 shows CNV lesion areas for all treatment groups. Although CNV isassumed to not be fully developed at Week 1, it is presented forcompleteness: A) No significant difference is seen between non-treatedanimals and drug-free DDS treated animals (“Blank DDS”) at any timepoint; B) ranibizumab-loaded DDS treated animals exhibited a significantdecrease in CNV lesion areas from Week 4-Week 12 compared to non-treatedanimals whereas bolus ranibizumab treated animals only had a significantdecrease in lesion area at Week 12; and C) bolus aflibercept treatedanimals had significantly smaller CNV lesions compared to non-treatedanimals at Week 4 and Week 12. However, aflibercept-loaded DDS treatedanimals had significantly smaller CNV lesions than bolus treated animalsfrom Week 8-Week 12 (p<0.05).

FIG. 15 shows representative composite FA images from Week 12 oftreatment groups: A) non-treatment, B) drug-free DDS, C) ranibizumabbolus, D) ranibizumab-loaded DDS, E) aflibercept bolus, and F)aflibercept-loaded DDS. As is seen, 5-6 CNV lesions were induced in eachtreatment group and the severity of the lesions were approximately thesame at Week 12.

DESCRIPTION OF THE INVENTION

The present invention provides a hydrogel delivery composition andmethod that can delivery treatment materials (e.g., drugs, etc.) in acontrolled matter for an extend period of time. Encapsulating andreleasing, for example, a bioactive drug for a long period of time is adifficult task. Many laboratories have tried to do this with limitedsuccess.

The composition of embodiments of this invention combines both hydrogeland microcapsules for treatment. Degradable microcapsules are suspendedin a degradable thermo-responsive hydrogel. The hydrogel isthermo-responsive at a physiological temperature of about 32° C. toabout 37° C., such as to change, via crosslinking, from a liquid-likestate at room temperature to a more solid state at body temperature.Exemplary hydrogel materials include poly(N-isopropylacrylamide),poly(latic acid), polysaccharide chitin, alginate, polyethylene glycol(PEG) and/or diacrylate (DA), or combinations or copolymers thereof.Exemplary copolymers include block copolymers PEG-PLLA-DA orPLA-PEG-PLA.

The thermo-responsive hydrogels of this invention provide an injectableplatform material that solidifies once injected into position. Smallgauge needles, e.g., about 20 to 33 gauge, and preferably about 25 to 27gauge, such as used in conventional intravitreal injection treatment,can be used to deliver the composition of this invention. This can be asignificant benefit as the injections can occur in doctor's officerather than in an operating room. The thermo-responsive hydrogels ofthis invention are injected in the “liquid-like” form and “solidify” toa gel material at body temperature. The transition time can becontrolled, so that the hydrogel can be used for various applications.

The hydrogels are desirably biodegradable, and can be either partiallyor fully biodegradable. The duration of degradation can also becontrolled. In embodiments of this invention, the degradation rate iscontrolled by the type and/or amount of crosslinker contained in the‘liquid’ form of the composition. In another embodiment, the degradationrate is modified by using different concentrations of and/or chaintransfer agents (e.g., glutathione) with poly(N-isopropylacrylamide).

In embodiments of this invention, the thermo-responsive hydrogel is usedto suspend or otherwise entrap microcapsules, e.g., nano- ormicrospheres, for easier delivery as well as localized delivery of themicrocapsules. Microcapsules generally will not stay in the eye wheninjected alone. Studies have shown that microcapsules will clear withinabout fifty days in normal eyes and within fourteen days invitrectomized eyes. Injecting microcapsules alone to deliver a long-termdrug treatment will not work. Another concern is that “free floating”microcapsules (if they are big enough) can interfere with visualfunction, in that a patient may ‘see’ microcapsules floating andinterfere with vision. By incorporating microcapsules into hydrogelaccording to this invention, the hydrogel structure ‘keeps’ themicrocapsules in place to provide longer-term delivery. Keepingmicrocapsules in the hydrogel also avoids interfering with the centralvisual pathway (visual perception).

The microcapsules are also desirably biodegradable, and can be eitherpartially or fully biodegradable. The duration of degradation and/orrelease can also be controlled. The degradation time and thus releaserate can be controlled, for example, through changing the polymer ratio,modifying the molecular weight of polymer(s), surface modification,and/or manufacture procedure. Currently preferred biodegradablemicrocapsules are based on poly(lactic-co-glycolic acid) (PLGA).Exemplary microcapsules according to this invention are formed using anysuitable microencapsulation process from poly(lactic-co-glycolic acid),poly(latic acid) (PLA), polysaccharide chitin, alginate(polysaccharide), or combinations or copolymers thereof. Exemplarycopolymers include, without limitation, PLGA-chitin and block copolymerswith polyethylene glycol (PEG) and/or diacrylate (DA), such as PLGA-PEG,PEG-PLLA-DA, PEG-PLGA-PEG, and PLGA-PEG-PLGA.

The invention includes a microencapsulated treatment agent suspended ina thermo-responsive hydrogel, for the purpose of delivering thetreatment agent to a delivery site. The microencapsulated treatmentagent-in-hydrogel composition of this invention changes to a second,more solid physicochemical state upon administration, and then degradesto release the treatment agent. The hydrogel keeps the microcapsule inplace (localized to delivery site) and prevents free movement ofmicrocapsule in and/or around the eye. The hydrogel can also act asanother release barrier and prolong the release time. The appliedmicrocapsules release the bioactive treatment agent over time afterapplication. Any suitable treatment agent can be encapsulated by anysuitable encapsulation method. Exemplary treatment agents include,without limitation, drugs, antibiotics (e.g., vancomycin, gentamicin),corticosteroids (e.g., dexamethasone, triamcinolone acetonide),anti-VEGF (e.g., pegaptanib, vevacizumab, ranibizumab, aflibercept,convercept), anti-PDGF, peptides, enzymes, biological agents (e.g.,nucleic acids: DNA, RNA), various stem or other cells, or combinationsthereof.

Treatment agent(s) can release from the microcapsule only, from both themicrocapsule and the hydrogel, from combinations of different sizemicrocapsules, from both different size of microcapsules and hydrogel,etc. Combination of treatment agents can be released (e.g., anti-VEGFand anti-PDGF, or combination of two different antibiotics) from thesame or different microcapsules and/or from the microcapsule and thehydrogel. These variations allow for different release rates and/ortreatments. In one embodiment of this invention, the thermo-responsivehydrogel is used to deliver a same or different agent in addition to themicrospheres. The hydrogel itself can be, for example, drug-loaded andthe release can be controlled; hence, providing another option todeliver agents in conjunction with microcapsules. This can be ideal in along-term delivery. For example, hydrogels can deliver “loading” dosesof drugs and the microspheres deliver “maintenance” doses.

Microcapsule-hydrogel platforms of this invention can provide long-termrelease of anti-VEGF, such as a controlled release of anti-VEGF for over6 months. Both in vitro and in vivo studies have shown that releasedanti-VEGF agents are active for the duration. This means that anti-VEGFcan be safely stored in the drug delivery platform of this invention andthe released drug has a positive effect clinically. Both modifiedmicrocapsule fabrication steps (adding protective agent for treatmentagent) and using hydrogel provided both extended release and bioactivityof the drug.

The delivery system can encapsulate various ocular drugs. The platformof this invention can replace monthly conventional intravitrealinjection treatment. Another novel application is to release multipledrugs at the same time. By encapsulating different drugs (or the samedrug with different dosages), drugs can be controlled and released atdifferent rates. As an example, clinically, there is growing evidencethat dual treatment of anti-VEGF and anti-PDGF (both deliveredconventionally intravitreal injection method) may be beneficial overmonotherapy of anti-VEGF. The platform of this invention can be utilizedto release both anti-VEGF and anti-PDGF for an extended period of time.

As discussed above the composition of this invention can be injected,and can also be used topically or in other medical procedures/devices.As an example, an illuminated microcatheter (such as iTrackmicrocatheter, iScience Interventional) can be used to deliver variousagents (encapsulated drugs or cells) into the suprachoridal space.Microcatheter injection has not typically been successful due to abackflush of drug after the injection. The biodegradablethermos-responsive composition of this invention will help keep thetreatment agent in place (due to the change from liquid-like tosolid-gel) after injection.

The composition of this invention can also be injected around the eyeglobe to act as a temporary scleral structure, such as a scleral bucklefor retinal detachment treatment. Currently, only permanent scleralbuckles (made out of surgical sponge or plastic) are used even thoughthe original intent of buckle use was a temporary procedure. Thecomposition can be injected directly into the globe to providestructural support (like a scleral buckle). Because the composition isbiodegradable, there will be no need to remove the hydrogel.Furthermore, it can be combined with agents (e.g., antibiotic) toprevent any post-procedure infection. Since it will be simple injection,the procedure should be minimally invasive. Because the polymercomposition can be controlled, the hydrogel can be made more firm togive sufficient structural support.

The composition can be used in existing or future medical devices. Forexample, the composition can be filled inside of ocular implants such asa Port Delivery System (PDS, developed by ForSight Vision4 and licensedby Genentech), which is a refillable, non-biodegradable implant(surgical procedure), or other implant reservoirs. Patients typicallyneed 4-5 refills over 12 months. Because the composition of thisinvention can release for over 6 months, a combination system can extendtreatment.

The present invention is described in further detail in connection withthe following examples which illustrate or simulate various aspectsinvolved in the practice of the invention. It is to be understood thatall changes that come within the spirit of the invention are desired tobe protected and thus the invention is not to be construed as limited bythese examples.

EXAMPLES

A drug delivery system (DDS) was prepared, composed of biodegradablepoly(lactic-co-glycolic acid) (PLGA) microspheres suspended within athermo-responsive, injectable poly(N-isopropylacrylamide)(PNIPAAm)-based hydrogel. In the initial development of this DDS,ovalbumin was used as a model protein to demonstrate controlled andextended release for approximately 200 days. By suspending themicrospheres within hydrogel, the initial burst (IB) was greatly reducedand release was extended by ˜30% compared to microspheres alone. Severalexcipients were incorporated into the DDS to protect against the acidicdegradation products of PLGA as well as to protect the protein (drug)during preparation, storage, and release. The objective of this work wasto demonstrate the capability of the microsphere-hydrogel DDS of thisinvention to release either ranibizumab or aflibercept in a controlledand extended manner and to determine whether the drugs remain bioactivethroughout release.

Ranibizumab and aflibercept were radiolabeled (to determine releaserate) with iodine-125 using iodination beads (Pierce, Rockford, Ill.,USA) and then dialyzed against double-deionized water (ddH₂O) using adialysis cassette (MWCO 2 kDa, Pierce) to remove unbound, free iodine.Labeled proteins were lyophilized, weighed, and dissolved in 1×phosphate-buffered saline (PBS, pH 7.4) to create stock solutions of 10mg/mL for each protein, which were stored at −80° C.

All subsequent chemicals were purchased from Sigma-Aldrich (St. Louis,Mo., USA). Each anti-VEGF was incorporated into PLGA microspheres usinga modified double-emulsion, solvent evaporation technique described inOsswald C. R. et al., Controlled and Extended Release of a Model Proteinfrom a Microsphere-Hydrogel Drug Delivery System, Ann Biomed Eng., Apr.3, 2015. Briefly, each protocol contained the same excipients in theinner aqueous (w₁) and oil (o) phases and each protocol was made intriplicate. In the w₁ phase, 12.5 mg bovine serum albumin (BSA), 10 mgPEG (molecular weight, MW, 8 kDa) (added to ‘protect’ the agent), and2.5 mg sucrose were dissolved in 100 μL of stock anti-VEGF solution. Inthe o phase, 125 mg PLGA 75:25 (4-15 kDa, acid terminated) and 3.75 mgMg(OH)₂ (also added to ‘protect’ the agent) were dissolved in 0.5 mLdichloromethane.

The primary water-in-oil emulsion (wt/o) was created by vortex at 3200rpm for 90 s (Fisher Scientific Analog Vortex Mixer; 120 V; speed 10×).The w₁/o emulsion was immediately added to the outer aqueous phase (w₂)containing 10% (w/v) polyvinyl alcohol (PVA). The secondary(water-in-oil)-in-water emulsion (w₁/o/w₂) was created by vortex at 2200rpm (speed 5×) for 90 s. The w₁/o/w₂ emulsion was then added to 75 mL of0.2% PVA followed by solvent evaporation on a stir plate (400 rpm for 3h). Microspheres were harvested by centrifugation (2000 ref), washedthree times in ddH2O, lyophilized to a dry powder, and stored at 4° C.

For each anti-VEGF agent, encapsulation efficiency (EE) was determinedfrom the radioactivity measured using a gamma counter (Cobra-IIAuto-Gamma, Packard Instrument Co., Meriden, Conn.) before and aftermicrosphere preparation. EE was defined as the percent-drug within themicrospheres relative to the theoretical loading amount.

To measure microsphere diameter, microspheres were fabricated asdescribed above with non-radiolabeled drug. A sample was suspended inddH₂O and imaged with a microscope (×20 objective, Carl Zeiss, Germany).Images of microspheres (n=135) were analyzed using ImageJ (developed byWayne Rasband, National Institutes of Health, Bethesda, Md.) to measurethe diameter.

Morphology of the microspheres was also examined by scanning electronmicroscopy (SEM). Dry microspheres were mounted on double-faced adhesivecarbon tape on metal stubs, sputter-coated with gold (E5000M S.E.M.Coater, Polaron Equipment Ltd., UK), and further analyzed in a scanningelectron microscope (JSM-5900LV, Jeol USA, Inc., Peabody, Mass.).

PNIPAAm-PEG-diacrylate (DA) hydrogels were synthesized using a methoddescribed in Osswald C. R. et al. Hydrogels were made in triplicate andprepared by dissolving PEG-DA (MW 575 Da, 2 mM), N-tert-butylacrylamide(47 mM), and ammonium persulfate (13 mM) in 1× Dulbecco's PBS (withCaCl₂ and MgCl₂). Then, NIPAAm (350 mM) was added to create the hydrogelprecursor. Microspheres (15 mg) were suspended in 1 mL of precursor in a2 cc microcentrifuge tube and kept on ice. Polymerization of thehydrogel was initiated by adding N,N,N′,N′-Tetramethylethylenediamine(168 mM). After polymerization, hydrogels were collected and washedthree times in ddH2O.

Release of anti-VEGF agents was determined using a separation methodalso described in Osswald C. R. et al. Briefly, 15 mg of microspheresand 1 mL, of the DDS (15 mg microspheres in 1 mL hydrogel) weresuspended in 1.5 mL of 1×PBS to ensure detectable levels of anti-VEGFconcentration throughout release. Release profiles were conducted at 37°C. under mild agitation and at predetermined intervals, 1 mL ofsupernatant was removed after a brief centrifugation and replaced withan equal volume of fresh buffer. Supernatants were read using a gammacounter (Packard). Cumulative release was calculated as a percent ofencapsulated drug and was considered complete when the microspheres hadcompletely degraded based on visual inspection. The IB was defined asdrug released within the first 24 hours.

For the bioactivity studies, non-radiolabeled release samples were keptat −80° C. until bioactivity testing. To determine bioactivity, humanumbilical vascular endothelial cells (HUVECs) were cultured,trypsinized, and seeded in 96-well plates at 5000 cells per well ingrowth medium (EGM-2, Lonza Inc., Allendale, N.J., USA). After the cellshad grown to ˜80% confluence (48 hr), cell growth was arrested bywashing the plates twice with 1×PBS and then adding 50 μl of basalgrowth media (EBM-2, Lonza) to each well. To achieve VEGF-inducedproliferation, 25 μL of 10 ng/mL exogenous VEGF (taken from a BulletKit,Lonza) and 25 μL of a release sample of anti-VEGF were then added toeach well. Each release sample was done in duplicate for a total of sixsamples per time point (microspheres alone and DDS made in triplicate).A solution containing a tetrazolium compound[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS] and an electron coupling reagent (phenazineethosulfate) (MTS assay, CellTiter 96® AQ_(ueous) One Solution Reagent;Promega, Madison, Wis., USA) was used according to the manufacturer'sprotocol to determine cell proliferation and cytotoxicity after two daysof exposure to the release samples. In the MTS assay, cell proliferationis proportional to the optical density (OD) of the sample when readusing a spectrophotometer at 490 nm from 1-4 hours. Cell proliferationwas normalized relative to those wells receiving only exogenous 10 ng/mLVEGF (negative control). Positive controls consisted of 25 μL of aclinical dose of drug (10 mg/mL ranibizumab or 40 mg/mL aflibercept)added to wells containing 10 ng/mL VEGF.

All values are reported as mean±standard error and in all graphs, errorbars represent standard error. Significant differences in microspherediameter, EE, and IB were determined using Student's t-test. For thebioactivity studies, significance at each time point was determinedusing Student's t-test. Significance between the test conditions wasdetermined using one-way ANOVA followed by the Holm-Sidak test. Unlessotherwise noted, significance represents p<0.05.

PLGA microspheres loaded with either ranibizumab or aflibercept weresuccessfully created and suspended within a thermo-responsivePNIPAAm-based hydrogel. Representative images of ranibizumab- andaflibercept-loaded microspheres can be seen in FIGS. 1A and 1C,respectively, with microspheres appearing smooth and non-porous. Afterthe microspheres were suspended within the hydrogel, the DDS remainedinjectable through a 28 G needle at room temperature (22° C.; FIGS. 1Band 1D). Particle size and EE for ranibizumab- and aflibercept-loadedmicrospheres alone and suspended in hydrogel can be seen in Table 1. TheEE was not significantly diflerent for either drug when comparingmicrospheres alone to those in hydrogel (p>0.65); however,ranibizumab-loaded microspheres had twice the EE compared toaflibercept-loaded microspheres.

TABLE 1 Characteristics of release for both ranibizumab- andaflibercept-loaded microspheres and the same suspended in hydrogel.Diameter Encapsulation Efficiency Initial Burst (μm) Percent (%) Weight(μg) Percent (%) Weight (μg) Ranibizumab Alone 7.5 ± 0.4 91.3 ± 2.5149.6 ± 3.7 50.3 ± 2.1 113.2 ± 4.7  in hydrogel 89.5 ± 2.8 106.2 ± 3.021.0 ± 2.0 22.2 ± 2.2 Aflibercept Alone 8.0 ± 0.3 44.8 ± 2.0 169.8 ± 3.483.3 ± 2.1 65.3 ± 0.7 in hydrogel 44.6 ± 1.4  65.4 ± 0.9 20.1 ± 0.8 31.1± 0.5

The hydrogel provided a significant reduction in the IB for bothanti-VEGF drugs. For ranibizumab, the IB was reduced by 58% (p=0.0003)and for aflibercept, the IB was reduced by 76% (p=0.0002) (Table 1).Beyond the first week of release, release from microspheres alone forboth ranibizumab and aflibercept was minimal (FIGS. 2A and 3A). Thisrelease phenomenon is often seen in protein-loaded microsphere. Itresults from the majority of protein being surface-bound, which quicklydiffuses away. Thus, there is minimal protein entrapped within the bulkof the microsphere, which precludes release at later time points frommicrospheres alone.

In contrast, suspending microspheres in hydrogel resulted in steadyrelease of 0.153 μg/day of ranibizumab (FIG. 2B) and 0.065 μg/day ofaflibercept (FIG. 3B) from Days 7-196. Thus, the hydrogel providessufficient resistance to the diffusion of surface-bound anti-VEGF, whichyields controlled release throughout. Additionally, both ranibizumab-and aflibercept-loaded microspheres completely degraded after 154 days,whereas suspending the microspheres in hydrogel extended the release ofboth drugs by 27.2% to 196 days.

Hydrogels were checked for translucency beginning on Day 154 (time atwhich microspheres alone had completely degraded but not microspheres inhydrogel) to determine whether the microspheres had degraded. This wasdone by lowering the temperature of the hydrogel from 37° C. to 22° C.and determining, by visual inspection, whether microspheres remainedwithin the hydrogel. The hydrogels returned to their translucent stateat 203 days; thus, degradation of the microspheres was determined to becomplete by 196 days. (11) An additional 25.1±12.1% (27.3 μg) ofranibizumab and 32.7±1.1% (21.4 μg) of aflibercept remained entrapped inthe hydrogel after complete degradation of the microspheres (FIGS. 2 and3, respectively; circled in red).

Bioactivity was maintained throughout release. A proportional increasein HUVEC proliferation was observed with increased VEGF concentration(FIGS. 4 and 5, left panels). Positive controls (clinical dose of eachdrug) significantly inhibited VEGF-induced proliferation of HUVECs(p<0.0007) and no difference was seen between positive controls andbasal growth medium (BGM, p>0.92).

For both drugs, significant inhibition of VEGF-induced HUVECproliferation was observed during the IB (p<0.05). At all subsequenttime points, HUVEC proliferation was less for the drug-loaded DDScompared to the non-loaded DDS counterparts (labeled as “Blank-gel” inFIGS. 4 and 5). For both the ranibizumab- (FIG. 4, right panel) andaflibercept-loaded DDS (FIG. 5, right panel), significant inhibition ofHUVEC proliferation was observed at many time points throughout release,including later release time points (p<0.05). Additionally, both drugsremain bioactive throughout release as indicated by a lower normalizedOD for drug-treated HUVECs compared to control at every time point(FIGS. 4 and 5, right panes). However, at various time points, aninsufficient amount of bioactive anti-VEGF was available tosignificantly inhibit HUVEC proliferation (p>0.05).

Controlled and extended release of bioactive ranibizumab and afliberceptwas achieved for ˜200 days by encapsulating these drugs in PLGA 75:25microspheres suspended in a PNIPAAm-based hydrogel. Compared tomicrospheres alone, the hydrogel significantly lowered the IB of bothdrugs and extended the release of each by almost a third. Additionally,the hydrogel allowed for significant bioactivity of both drugs to bemaintained throughout. If these results are verified in vivo, bioactiveand controlled release for nearly 200 days is a significant step towardsreplacing the current monthly/bimonthly treatment regimens approved forthese drugs.

Interestingly, ranibizumab-loaded microspheres had twice the EE ofaflibercept-loaded microspheres even though the theoretical loading wasthe same and the drugs were treated identically in the fabrication ofthe microspheres. A likely cause for this is the difference in MWbetween the drugs. It has been shown across a variety of fabricationtechniques, including the double-emulsion technique used in this study,that smaller proteins tend to have a higher EE. Larger proteins tend toremain surface-bound at the polymer-water interface, as was evidenced bythe significantly higher IB for aflibercept (MW 115 kDa) compared toranibizumab (MW 48 kDa). Additionally, charge and tensioactiveproperties of the proteins may have contributed to the differences seenin EE.

Suspending microspheres within the thermo-responsive hydrogel accordingto this invention significantly reduced the IB by 58% and 76% forranibizumab and aflibercept, respectively. The reduction in IB may beattributed to the fabrication of hydrogel (e.g., surface diffusion ofdrug into hydrogel precursor prior to gelation) and the hydrogel actingas a diffusion barrier. A larger protein would see a greater diffusionbarrier; thus, it was anticipated and shown that the reduction in IB wasgreater for aflibercept than ranibizumab. Importantly, suspending themicrospheres within the thermo-responsive hydrogel yielded steadier andmore controlled release compared to microspheres alone. Thismicrosphere-hydrogel DDS approach has been shown previously to providebetter control and extended release compared to microspheres alone.Thus, the DDS of this invention has two potential benefits for thetreatment of posterior segment eye diseases: it would prevent theindependent movement of microspheres within the eye as well as provideprolonged and controlled release of anti-VEGFs.

For both anti-VEGFs, a residual amount of drug was observed trapped inthe hydrogel at the end of the study (final time point, FIGS. 2 and 3).In a study by one of the inventors using PNIPAAm-based hydrogel toencapsulate BSA and immunoglobulin G (IgG), a similar entrapment wasobserved. Aflibercept had a higher residual entrapment (32.7±1.1%) thanthe smaller ranibizumab (25.1±12.1%), which had a higher residualentrapment than the smaller ovalbumin (45 kDa) observed in our priorstudy (8.01±0.76%). Non-degradable hydrogels were used intentionally inthis study to simplify the experimental factors and focus on the releaseof ranibizumab and aflibercept. The issue of entrapped drug using thehydrogel can be mediated by utilizing fully biodegradable hydrogels.Hydrogels with varying lengths of degradation can have an effect onrelease from microspheres. For example, FIG. 6 summarizes data from anexperiment (though 180 days) showing release of ranibizumab frombiodegradable microspheres and a biodegradable thermo-responsivehydrogel. Different amounts of crosslinker (PLLA-PEG-DA) were used tochange the degradation rate.

Including the residual entrapped drug, incomplete release (that is, notachieving 100% cumulative release) was still observed. This phenomenonis frequently reported in many protein-loaded PLGA microsphere deliveryplatforms and may be due to protein instability. Protein aggregation andnon-specific protein adsorption are typically the leading causes ofincomplete release and differences in the isoelectric point (pI) of eachdrug may be to blame. At pH lower than the pI, proteins carry a netpositive charge and vice versa. In a previous study using ovalbumin,complete release was observed. The pI of ovalbumin is ˜4.6, whereasmonoclonal antibodies (e.g., ranibizumab) typically have a pI of ˜8.Thus, performing these experiments at physiological pH (7.4) may haveinfluenced the release due to possible charge interactions between thedrugs, PLGA, and hydrogel.

Significant bioactivity is seen during late release time points (i.e.,beyond 100 days), a timeframe well beyond the IB where a large amount ofdrug was released. In our initial characterization of this DDS withovalbumin, a large second burst occurred after Day 70 and release beyondthe second burst occurred at a greater rate than prior to the secondburst. The IB can be readily explained by diffusion of drug located nearthe particle surface through short diffusion pathways. As PLGA swellsand degrades, the pores become large enough to allow for the release ofentrapped protein, which can lead to a second burst. Although a largesecond burst was not observed in this study for either anti-VEGF-loadedDDS, it is likely that at these later time points, even though theamount of protein was small, the protein was likely intact and thus asbioactive as during the IB. Proteins at interfaces (e.g., surface-boundprotein during the IB) tend to easily denature whereas proteinsentrapped within the microsphere (i.e., release at later time points)tend to remain intact and bioactive.

The release of bioactive anti-VEGF for over six months is a significantstep toward eliminating monthly or bimonthly bolus injections of drugsneeded to treat many posterior segment eye diseases. The extended andcontrolled release of anti-VEGF agents that the DDS of this inventiondemonstrates may be a significant advancement in the delivery of proteintherapeutics to the eye once validated in vivo. The DDS of thisinvention has the potential to improve upon the socio-economic impactassociated with recurrent injections and to lower the risk forassociated potential complications.

To further validate the potential of the DDS of this invention to treatposterior segment eye diseases, the in vivo efficacy of the DDS wasdetermined using a rat model of CNV. Animal models of CNV fall intothree categories: laser and light induced, surgically induced, andtransgenic and knockout mouse models. These models have a commonbeginning: a break or defect in Bruch's membrane. Development of CNV isa dynamic process with initiation, maintenance, and involution stages.Currently, the most common way to induce CNV in animals is through theuse of laser photocoagulation as it is relatively simple to create, isinexpensive, and is reproducible.

In rats, approximately 75% of lesions are vascularized within the firstweek following CNV induction; after ten days, CNV is fully developed andremains so for at least 35 days after induction. Additionally, it hasbeen found that the highest rate of blood vessel growth (3-10 days afterinduction) correlates with the peak expression of VEGF and its receptor.Furthermore, using vascular cast images it has been shown that featuresof laser-induced CNV in rats were similar at one and three months butthat CNV had atrophied by six months. Accordingly, lesions should bemonitored at least weekly in the beginning of CNV development, with theassumption that CNV is not fully developed until two weekspost-induction and lesions may begin to atrophy after three months.

Several methods exist to quantify CNV lesion growth. Often, a four-tiergrading scale is used ranging from no leakage to severeleakage/hyperfluorescence, where FA images are analyzed by trainedretinal specialists, averaged, and compared. However, this methodremains somewhat subjective and can be influenced by image properties(e.g., brightness and contrast). More complex and objective methodsinvolve flat-mount preparations, immunohistochemistry with serialreproductions, and OCT. However, these complex methods can quicklybecome quite expensive and time consuming. To address the issues ofobjectivity, ease of use, and cost-effectiveness, a novel technique wasdeveloped based on FA images and Otsu thresholding to monitor CNV lesiongrowth.

Otsu thresholding allows for the automatic reduction of a grayscaleimage into a binary foreground/background image such that an optimumthreshold separating the two classes maximizes the inter-class varianceof pixel intensity. This can be extended to separate intermediate levelsand has been implemented as a plug-in for the open-source softwareImageJ (NIH). To quantify CNV areas, three levels (or “regions”) aredefined: background, diffuse leakage, and CNV (FIG. 7). When determiningCNV areas, the CNV lesion is outlined with blood vessels avoided. Thelesion area is measured in pixels, which can then be converted into areausing a scaling factor.

The ability of controlled and extended release of anti-VEGF from the DDSto inhibit CNV lesion growth was tested and compared to that of acorresponding bolus injection of anti-VEGF. Six treatment groups wereobserved: non-treatment, drug-free DDS, ranibizumab-loaded DDS,ranibizumab bolus, aflibercept-loaded DDS, and aflibercept bolus. Alongwith determining CNV areas, ocular health was monitored to demonstratethat the DDS was biocompatible and well-tolerated in vivo. It washypothesized that controlled and extended release of anti-VEGF from theDDS will more effectively treats CNV than a bolus injection and thatocular health will be maintained throughout the study.

All animal procedures were in accordance with protocols approved by theInstitutional Animal Care and Use Committee at the Illinois Institute ofTechnology, and with the principles embodied in the statement on the useof animals in ophthalmic and vision research adopted by the Associationfor Research in Vision and Ophthalmology. Long-Evans male rats (1-3months, 300-350 g) were purchased from Harlan Laboratories(Indianapolis, Ind., USA). Animals were anesthetized using 80 mg/kg ofketamine hydrochloride (Fort Dodge Animal Health, Fort Dodge, Iowa, USA)and 10 mg/kg xylazine (AnaSed® Injection, Akorn, Inc., Decatur, Ill.,USA) via intraperitoneal (IP) injection. Proparacaine drops (Bausch andLomb, Rochester, N.Y., USA) were used to anesthetize the corneasthroughout the procedure and pupils were dilated using phenylephrine(Bausch and Lomb) and atropine drops (Bausch and Lomb). Heart rate andblood oxygen saturation were monitored with a PulseOximeter (8500AV;Nonin Medical Inc., Plymouth, Minn., USA). Animals were placed on acustom-built heated stage and monitored to maintain a core bodytemperature of 37° C.

Non-radiolabelled, ranibizumab-, and aflibercept-loaded microsphereswere prepared. Fifteen (15) mg of microspheres were put in a 2 ccmicrocentrifuge tube and placed under UV light for 30 min to sterilize.Under sterile conditions, the hydrogel precursor and initiator weresterile-filtered using 13 mm syringe filter (0.22 μm, Fisherbrand,Thermo Fisher Scientific, Waltham, Mass., USA). One (1) mL of hydrogelprecursor was added to the microspheres and the tube was invertedseveral times to suspend the microspheres. The initiator was then addedand free radical polymerization occurred on ice for 30 min. Hydrogelswere washed three times in sterile PBS, loaded into 0.5 cc U-100 insulinsyringes (28G½; Becton Dickinson & Co., Franklin Lakes, N.J., USA), andstored at 4° C.

Laser photocoagulation was performed using an argon-green laser(AKC-8000, NIDEK, Inc., Fremont, Calif., USA) attached to a slit lampwith a laser power of 400 mW, duration of 100 ms, and spot diameter of50 μm. Using a 90-diopter lens, the posterior pole of the eye was viewedand the laser beam focused on the retina. Five to six lesions per eyewere induced two to three disc diameters from, and centered on, theoptic disc. Laser-induced disruption of Bruch's membrane was identifiedby the appearance of a bubble at the site of photocoagulation. Laserspots that did not result in the formation of a bubble were excludedfrom study.

Animals were separated into six groups to determine the ability of theDDS of this invention to treat CNV. For analysis purposes, lesions wereconsidered independent within each treatment group and each groupcontained two rats for a total of four eyes and up to 24 lesions pertreatment group. The treatment groups were as follows: 1) control group,which did not receive any treatment or injection after laser induction(“non-treatment”); 2) non-loaded DDS (“Blank-gel”); 3)ranibizumab-loaded DDS (“Rani.-gel”); a single bolus injection ofranibizumab at clinical dose (10 mg/mL, “Rani.”); a single bolusinjection of aflibercept-loaded DDS (“Aflib.-gel”); aflibercept atclinical dose (40 mg/mL, “Aflib.”). All IVT injections were 5 μL andperformed immediately after CNV induction.

The corneal electroretinogram (ERG) is a non-invasive measurement thatrepresents the overall electrical activity of the retina in response toa stimulus flash. It is routinely used to assess retinal cellularactivity and in toxicology studies. Each component of the ERG can beused to assess different retinal cell types and any alterations in thefunction of the retina due to the DDS would appear as changes inamplitude or sensitivity of various ERG components. Under scotopicconditions, the ERG has two important components: the a-wave and b-wave.The rising edge of the negative a-wave is generated by thephotoreceptors in the outer retina; the positive b-wave is generatedprimarily by the bipolar cells and/or Müller cells. Another component ofthe ERG is the c-wave, which originates in the RPE and can be used toassess the functional integrity of the photoreceptors, the RPE cells,and the interactions between them; however, for the purposes of thisresearch, the c-wave was not recorded or analyzed.

Under dim red light prior to and at each time point after CNV inductionand DDS injection, ERG experiments were performed under scotopicconditions, with animals dark-adapted overnight prior to the experiment.Electroretinograms, in response to the full-field Ganzfeld stimulation,were recorded by a gold wire loop placed on the cornea. The referenceand ground electrodes were 30 G platinum subdermal needle electrodes(SAFELEAD™ F-E2, Grass Products, Natus Neurology, Warwick, R.I., USA)inserted into the cheek and nape of the neck, respectively. A-wave andb-wave intensity responses were recorded by presenting single flashes ofincreasing intensity (5×10⁻⁴ to 305.7 sc cd·s·m⁻²) and allowing the eyeto dark adapt for 1 min after each flash. The intensity-response valuesfor the a-wave and b-wave were fit with the Naka-Rushton equation, todetermine a half-saturation intensity of the responses and maximalresponse.

Prior to treatment, post-treatment, and weekly thereafter, intraocularpressure (IOP) measurements were taken using an applanation tonometer(TONO-PEN®XL, Medtronic, Minneapolis, Minn., USA). The tono-pen has beenshown to effectively measure rat IOP. It should be noted that generalanesthesia, including combined ketamine and xylazine, has been shown toinduce rapid and substantial decreases in IOP as well as increasedinter-animal variability in IOPs.

A confocal scanning laser ophthalmoscope (cSLO) system (HeidelbergRetina Angiograph (HRA), Heidelberg Engineering, Heidelberg, Germany)was used to image the retina. FA still images were captured at 1-2 min,10 min and 20 min after IP injection of 0.5 mL of 20% fluorescein dye(Sigma Aldrich, St. Louis, Mo., USA) and were used for quantification ofCNV lesion areas. All data was acquired prior to treatment and at 1, 2,4, 8, and 12 weeks post-injection.

CNV lesion areas were quantified using late-phase FA images. The use oflate-phase (20 min) images allows time for the fluorescein to clear thenormal vasculature, preventing vessel hyperfluorescence from interferingwith area measurements. Additionally, only images that were centered onthe lesion, well-focused on the choroid, non-saturated, and had evenillumination were used to quantify CNV.

An example of image processing using ImageJ is presented in FIG. 7. Themulti-Otsu thresholding (MOT) plugin for ImageJ was set to three levels,or “regions,” defined as: background (FIG. 7B), diffuse leakage(non-hyperfluorescent leakage; FIG. 7C), and CNV (hyperfluorescentleakage; FIG. 7D). The threshold value within ImageJ is then adjusted toinclude all pixels within the “Region 2” (CNV) plug-in output. The CNVlesion was then outlined by the user, being careful to avoid vessels andother non-lesion pixels (FIG. 7E). The measured number of pixels wasthen converted into area using a scaling factor based on the opticalproperties of the rat eye and field of view of the image using theequation:

${SF} = {\frac{\pi}{180} \times N^{\prime}F^{\prime} \times \frac{FOV}{RES}}$where SF is the scaling factor in square micrometers per pixel, N′F′ isthe distance from the second nodal point of the eye to the retina inmicrometers, FOV is the field of view in degrees, and RES is the lengthof the image in pixels. For the Long-Evans rats used in this study, SFwas 3.35 μm². Note that within ImageJ, the MOT plugin requires an 8-bitgrayscale image and measurements must be set to “Limit to threshold”under the “Analyze>Set measurements . . . ” tab, otherwise all pixelswithin the outline will be counted rather than only within the lesion.

The parameter values obtained from the Naka-Rushton analyses and IOPmeasurements were compared using the paired t-test verses control. Todetermine significant differences in CNV lesion areas, one-way ANOVA wasperformed and all pairwise multiple comparisons were done using theHolm-Sidak method. Unless otherwise noted, significance representsp<0.05. All values are reported as sample mean±standard error of themean.

In a previous, thorough evaluation of non-drug-loaded thermo-responsivehydrogel, no adverse effects were observed out to four weeks post-IVTinjection. As the microsphere-hydrogel DDS was to be evaluated over thecourse of 12 weeks, an additional ocular health and safety evaluationwas performed including ERG analysis and IOP measurements.

The outer retina was evaluated by the a-wave, which reflects theactivity of the photoreceptors. The sensitivity of the a-wave wasexamined through comparison of parameters obtained from the Naka-Rushtonanalyses and the intensity-response of the a-wave was determined before(control) and after injection of the DDS and the bolus drugcounterparts. As was expected, no significant differences were seen inmaximal a-wave response (control: 543.1±51.7 mV) or half-saturationintensity (control: 2.9±1.6 sc cd·s·m²) of non-treated animals (FIG. 8,top). Additionally, no significant differences in a-wave parameters wereseen for drug-free DDS treated animals (control Amax: 400.9±23.7 mV,control σA: 2.6±0.8 sc cd·s·m⁻²; FIG. 9, top). For both theranibizumab-loaded DDS (control Amax: 464.1±19.9 mV, control σA: 2.2±0.5sc cd·s·m⁻²) and bolus ranibizumab (control Amax: 419.9±24.0 mV, controlσA: 2.6±0.8 sc cd·s·m⁻²) treated animals, no significant differenceswere seen in a-wave parameters (FIGS. 10 and 11, top; respectively).However, for both the aflibercept-loaded DDS and bolus aflibercepttreated animals, a significant difference in maximal a-wave response isseen between control and Week 1-Week 12 (p<0.05). For theaflibercept-loaded DDS treated animals, the maximal a-wave responsepost-treatment was ˜20% lower than control (Amax: 516.0 51.1 mV) at alltime-points post-treatment (FIG. 12, top) and for the bolus aflibercepttreated animals, it was ˜40% (control Amax: 616.4±25.4 mV; FIG. 13,top). No significant difference in half-saturation intensity wasobserved for either aflibercept-loaded DDS (control σA: 4.5±2.5 sccd·s·m⁻²) or bolus aflibercept (control σA: 4.6±0.9 sc cd·s·m⁻²) treatedanimals.

Inner retinal function was evaluated by the b-wave, which reflects theactivity of the bipolar cells and/or Müller cells. As with the a-wave,the sensitivity of the b-wave was examined through comparison ofparameters obtained from the Naka-Rushton analyses and theintensity-response of the b-wave was determined before (control) andafter injection of the DDS and the bolus drug counterparts. As expected,no significant differences were seen in maximal b-wave response(control: 748.5±70.7 mV) or half-saturation intensity (control σB:2.6×10-3±8.0×10-4 sc cd·s·m⁻²) of non-treated animals (FIG. 8, bottom).A significant increase of ˜35% in maximal b-wave response was seen onlyat Week 12 compared to control for drug-free DDS treated animals(control B max: 684.7±37.8 mV; FIG. 9, bottom; p<0.05), suggesting thatthe DDS may cause a greater depolarization of the ON-bipolar cells ofthe retina in the long-term. Similar to the a-wave, for both theranibizumab-loaded DDS (control Bmax: 987.9±91.0 mV, control σB:3.3×10⁻³±1.0×10⁻³ sc cd·s·m⁻²) and bolus ranibizumab treated animals(control Bmax: 734.3±65.0 mV, control σB: 4.1×10⁻³±1.1×10⁻³ sccd·s·m⁻²), no significant differences were seen in b-wave parametersafter treatment (FIGS. 10 and 11, bottom; respectively). For theaflibercept-loaded DDS treated animals, no significant differences inb-wave parameters were observed (control Bmax: 790.4±51.8 mV, controlσB: 3.0×10⁻³±6.0×10⁻⁴ sc cd·s·m⁻²; FIG. 12, bottom). Surprisingly, forthe bolus aflibercept treated animals, a significant decrease in maximalb-wave response is seen post-treatment through Week 12, with the maximalb-wave response being ˜45% lower than controls (control Bmax:1236.1±121.5 mV, control σB: 4.6×10⁻³±1.4×10⁻³ sc cd·s·m⁻²; FIG. 13,bottom). Although a significant decrease in maximal b-wave response wasseen in these animals after treatment, control maximal a- and b-waveresponses for these animals were >50% higher than all other controlmeasurements, which may suggest abnormal experimental conditions duringcontrol ERG measurements (e.g., residual eye drops enhancing the contactof the cornea electrode or the position of the eye being closer to theflash stimulus than normal).

IOP measurements were recorded prior to and immediately after IVTinjection and at every time point thereafter. Table 2 shows the averageIOP measurements for all treatment groups at each time point. Theaverage control IOP values for all animals was 18.8±0.1 mmHg. As wasanticipated, a significant increase of ˜37% in IOP is seen immediatelyafter IVT injection in all treated animals (p<0.05). Increased IOPremained at Week 1 for the aflibercept-loaded DDS and both bolus treatedanimals (23.3±1.0 and 25.0±1.8 mmHg, respectively). By Week 2, onlybolus aflibercept treated animals had increased IOP (25.0±1.8 mmHg).Interestingly, at Weeks 8-12, the ranibizumab-loaded DDS treated animalshad significantly lower IOP verses control (17.0±0.8 and 17.3±1.0 mmHg,respectively) and at Week 12, bolus ranibizumab treated animals also hadsignificantly decreased IOP (17.3±1.5 mmHg). It should be noted that allIOP values were well within the range of what is normal in rats (15-25mmHg).

TABLE 2 Average intraocular pressure measurements for all treatmentgroups Treatment Control Post-Injection Week 1 Week 2 Week 4 Week 8 Week12 Non- 17.3 ± 2.1 — 22.0 ± 4.0 19.3 ± 0.6 19.0 ± 0.0 19.7 ± 0.6 17.3 ±1.2 treatment Drug-free 19.0 ± 2.4 25.5 ± 4.4* 21.8 ± 1.3 21.8 ± 1.521.3 ± 1.0 19.0 ± 0.8 18.8 ± 1.5 DDS Rani.-gel 19.5 ± 1.3 27.0 ± 0.8*21.5 ± 1.7 20.8 ± 1.3 20.8 ± 1.0  17.0 ± 0.8*  17.3 ± 1.0* Rani.-bolus20.0 ± 2.2 26.5 ± 1.3* 24.3 ± 1.7 20.3 ± 2.2 20.0 ± 2.0 19.8 ± 1.7  17.3± 1.5* Aflib.-gel 18.5 ± 1.3 25.0 ± 3.6*  23.3 ± 1.0* 20.8 ± 1.0 18.5 ±2.1 19.8 ± 1.0 18.3 ± 1.3 Aflib.-bolus 18.0 ± 1.4 26.5 ± 1.7*  25.0 ±1.8*  20.8 ± 1.0* 18.5 ± 1.3 19.3 ± 2.2 17.8 ± 1.5 *Significantdifferences in IOP compared to each treatments' control measurement (p <0.05)

The MOT technique effectively delineated the CNV lesion from thesurrounding retina in the experimental laser model, allowing forobjective quantification of CNV lesion areas. The CNV lesion areas forall treatment groups can be seen in FIG. 14. Representative composite FAimages show that the severity of CNV lesions at Week 12 for eachtreatment group was approximately the same (FIG. 15). Although CNV isassumed to not be fully developed at Week 1, it has been presented forcompleteness. As anticipated, no significant difference was observedbetween non-treated animals and drug-free DDS treated animals at anytime point. From Week 4-Week 12, the ranibizumab-loaded DDS hadsignificantly smaller lesion areas than non-treated animals and by Week12, these treated animals had CNV lesion areas that were 60% smaller(p<0.0002). Additionally, beginning on Week 1, ranibizumab-loaded DDStreated animals exhibited smaller CNV lesion areas than bolusranibizumab treated animals, however this difference was notsignificant. Furthermore, bolus ranibizumab treated animals only had asignificant decrease in lesion area at Week 12. Bolus aflibercepttreated animals had significantly smaller CNV lesions compared tonon-treated animals and aflibercept-loaded DDS at Week 1, however it isunknown whether this difference is meaningful as CNV growth is not fullydeveloped until ˜10 days. Additionally, bolus aflibercept treatedanimals had significantly smaller CNV lesions compared to non-treatedanimals at Week 4 and Week 12. However, by Week 8, aflibercept-loadedDDS treated animals had significantly smaller CNV lesions than bolus andDDS treated animals from Week 8-Week 12 (p<0.05).

For both anti-VEGF-loaded DDSs, it is important to note that significantdecreases in CNV lesion growth were achieved that exceeded that of bolusadministration in the long term. With a 5 μL bolus injection of aclinical dose of either anti-VEGF, 50 μg of ranibizumab or 200 μg ofaflibercept was delivered. In comparison, over the course of 12 weeksonly ˜450 ng of ranibizumab or ˜200 ng aflibercept was delivered fromthe DDS of this invention. Thus, there is great potential of controlledand extended release from the DDS of this invention in the treatment ofCNV.

The efficacy of the DDS to treat laser-induced CNV in rats was evaluatedin addition to determining the safety and biocompatibility of the DDS.Retinal cellular function was evaluated using ERG parameters determinedby fitting the intensity-response curve to the Naka-Rushton equation.Intraocular pressure measurements were also taken. Finally, CNV lesionareas were measured at every time point to determine whether the DDSeffectively treated laser-induced CNV in Long-Evans rats.

In animals treated with the drug-free DDS, photoreceptor cellularfunction was maintained and normal ERG activity was observed throughoutthe 12 weeks post-treatment. This result expands the time frame forwhich the thermo-responsive hydrogel DDS has been shown to cause nolong-term physiological effects on the outer retina. In a previousstudy, the thermo-responsive hydrogel was evaluated for four weeks andit did not cause any long-term physiological effects on thephotoreceptors. The lack of change in the outer retina was notsurprising as the photoreceptor cells are located farthest from thehydrogel injection. It follows, then, that the inner retinal cells aremore likely to experience any adverse effects due to the hydrogelinjection, and changes in inner retinal function would be apparent inthe b-wave. As was shown, inner retinal health and activity wasmaintained through Week 8. Interestingly, a ˜35% increase in maximalb-wave response was observed at Week 12 in all animals, which couldimply a meaningful adverse effect in long-term biocompatibility.However, no significant changes in a- or b-wave half-saturationintensity, which indicates the sensitivity of the retina toillumination, were seen in any treatment group throughout the entirestudy. The human rod system requires an intensity change of greater than14% for an observer to perceive a change in light intensity. Thus, whileno change in half-maximal sensitivity was observed, it is possible thatthe large increase in maximal b-wave response observed causedperceptible changes in the rodent's vision whereby the rat was moresensitive to light at Week 12 compared to control.

For both the bolus ranibizumab and ranibizumab-loaded DDS treatedanimals, no significant differences were seen in either maximal a- andb-wave response or half-maximal a- and b-wave response, furtherindicating that the DDS does not induce adverse effects on retinalactivity. Although no changes were observed in maximal b-waveparameters, a small by significant decrease (˜10%) was observed inaflibercept-loaded DDS treated animals after treatment. For reasonsmentioned above, this slight decrease would likely not cause perceptiblechanges in the rodent's vision. More surprising was the large (˜40%),sustained decrease in maximal a- and b-wave responses for bolusaflibercept treated animals subsequent post-treatment. These bolustreated animals had a similar number and severity of lesions as theother treated groups (FIG. 14), which eliminates the possibility thatthe decrease in the full-field ERG maximal a- and b-wave responses wasdue to greater CNV area (i.e., decreased retinal activity). However,control measurements for these animals were >50% higher than all othercontrol measurements, which may suggest abnormal experimental conditionsduring control ERG measurements (e.g., residual eye drops enhancing thecontact of the cornea electrode or the position of the eye being closerto the flash stimulus than normal). In the future, histologicalevaluation of the enucleated eyes will confirm whether this sustaineddecrease in maximal a- and b-wave response was due to irreversible toxicretinal damage.

The clinical dose of aflibercept (50 μL injection of 40 mg/mLaflibercept) when injected into the human vitreous (3.8 mL) yields avitreal concentration of 0.52 mg/mL. In this study, 5 μL of 40 mg/mLaflibercept was injected into the rat vitreous (13.6 μL vitreousvolume), which translates into a vitreal concentration of 10.8 mg/mL,which is a much higher vitreal concentration than that used in humans.In a recent ex vivo study on the electrophysiological toxicity testingof aflibercept in bovine retina, a significant decrease (43.1%) in themaximal scotopic b-wave response was observed directly after exposure to0.1 mg/mL aflibercept. After washing away the aflibercept, the b-waveresponse was no longer significantly decreased. So, it is possible thatfor rats, the high bolus aflibercept concentration used in this studycaused irreversible toxic retinal damage. However, in a study inchinchilla rabbits, a clinical dose of aflibercept was administered andwith a vitreous volume of approximately 200-300 μL, this translates intoa vitreal concentration of 6.67-10 mg/mL. It was found that this highvitreal concentration did not alter any ERG parameters at 24 hr or 7days after treatment; however, rabbits were only dark-adapted for 30minutes prior to ERG recordings and this is an insufficient amount oftime for complete dark-adaptation to occur. Importantly, the decreasedmaximal a- and b-wave responses in bolus aflibercept treated animals wasmediated by using the DDS as aflibercept is delivered slowly over timeand at much lower concentrations (i.e., two orders of magnitude less)and no changes in ERG parameters were observed in aflibercept-loaded DDStreated animals.

Retinal function can also be affected by changes in IOP. In the currentstudy, there was a significant increase in comparison to control in TOPmeasurements immediately after the injection, which resolved by Week 2for all treatment groups. This indicates that the IVT injection of theDDS of this invention had minimal impact on IOP. Surprisingly, in thebolus ranibizumab and ranibizumab-loaded DDS treated animals, asignificant decrease in IOP was observed at later time points. However,all values obtained in this study are in agreement with the normal rangeof IOPs for Long-Evans rodents. Although the adult rat vitreous volumeis ˜14 μL, IVT injection volumes of up to 5 μL have been shown by Dureauet al. (2001) to have good reproducibility with minimal loss of injectedsolution. Therefore, any changes seen in the ERG parameters were likelynot the result of altered IOP. Based on the ERG parameter analysis ofthe drug-free DDS and the IOP values for all treatment groups, it isbelieved that the DDS is safe, biocompatible, and well-tolerated.

No significant differences were seen between non-treated and drug-freeDDS animals at any time point. This suggests that the significantdecreases in CNV lesion areas observed in bolus or anti-VEGF-loaded DDStreated animals were in fact related to the anti-VEGF treatment. It hasbeen demonstrated that much less drug delivered constantly yields abetter treatment outcome than a bolus injection in the long term.Ranibizumab-loaded DDS treated animals exhibited a significant decreasein CNV lesion areas from Week 4-Week 12 compared to non-treated animalswhereas bolus ranibizumab treated animals only had a significantdecrease in lesion area at Week 12. Due to the short half-life ofranibizumab (t1/2=9 days), after 12 weeks, the ranibizumab that wasdelivered via bolus injection was effectively gone. It is important tonote that ranibizumab was administered immediately after CNV inductionand that a sufficiently high vitreal concentration of ranibizumab dosepresent during the highest rate of blood vessel growth (3-10 days afterinduction), which correlates with the peak expression of VEGF and itsreceptor. This may imply that the large initial bolus ranibizumab doseprevented CNV from fully forming and thus allowed CNV lesion areas tosignificantly decrease by Week 12.

Bolus aflibercept treated animals had significantly smaller CNV lesionscompared to non-treated animals at Week 4 and Week 12. However,aflibercept-loaded DDS treated animals had significantly smaller CNVlesions than bolus treated animals from Week 8-Week 12. Thus, theaflibercept-loaded DDS had greater efficacy in the long-term.Aflibercept is a more potent anti-VEGF than ranibizumab as demonstratedclinically by the need to administer ranibizumab more often thanaflibercept to achieve a similar treatment outcome. This may explain theearly advantage of bolus aflibercept compared to the DDS, yet extendeddelivery of aflibercept significantly reduced CNV lesion areas eventhough the rate of release for aflibercept from the DDS was 2.3× lessthan that of ranibizumab.

Interestingly, both anti-VEGF-loaded DDSs had roughly the same decreasedCNV lesion areas at Week 12, with aflibercept-loaded DDS areas only 2%larger than the ranibizumab-loaded DDS areas. In our preliminary studyon extended release of dexamethasone, a potent corticosteroid, from theDDS of this invention (nanospheres rather than microspheres), it hasbeen demonstrated that extended release of dexamethasone from the DDS ofthis invention greatly reduced CNV lesions areas compared to bolusadministration while delivering less than 5% of the amount of bolusdexamethasone. Together with the results presented here, this suggeststhat the efficacy of a variety of antiangiogenic agents could be greatlyimproved by controlled and extended release from the DDS of thisinvention.

There is debate as to whether bevacizumab, a humanized variant of theanti-human VEGF-A monoclonal antibody A.4.6.1, and ranibizumab, ahumanized monoclonal antibody and derivative of bevacizumab, areeffective in treating murine neovascularization. In a study evaluatingthe efficacy and safety of intravitreal injections of bevacizumab andranibizumab in the treatment of CNV in a rat model, no therapeuticeffect was observed out to 28 days over a range of doses both above andbelow the clinical dose of each drug. However, in several studies on theeffect of using bevacizumab to treatment corneal neovascularization,humanized anti-VEGF was found to be effective in the rat. It has beenshown that systemic or topical application of bevacizumab significantlyinhibited inflammation-induced angiogenesis in the cornea of mice andthat bevacizumab binds to mouse VEGF-A, which was determined by Westernblot, enzyme-linked immunosorbent assay (ELISA), and plasmon resonanceassay. Additionally, it has been demonstrated that topical bevacizumablimited corneal neovascularization in rats it has been found thatsub-conjunctival injection of bevacizumab can significantly inhibitcorneal angiogenesis. Furthermore, the results presented above clearlydemonstrate the efficacy of ranibizumab in treating murine CNV.

In summary, controlled and extended release of anti-VEGFs from the DDSwas shown to be more efficacious than bolus anti-VEGF treated animals ina laser-induced CNV murine model. Both anti-VEGF-loaded DDSs exhibitedvery similar efficacy at Week 12, suggesting that improved efficacy mayextend to other antiangiogenic agents. Additionally, the DDS was shownto be safe, biocompatible, and well-tolerated, with no long-term adverseeffects observed in drug-free DDS treated animals.

Thus, the invention provides a new and improved method to deliveranti-VEGF or other treatment agent for an extended period of time.Currently, there are over 4 million intravitreal injections done monthlyin U.S. alone to treat AMD, DR and other vascular disease. Limitinginjection to every 6 months will have a great socioeconomic impact aswell as reduce potential side effects of monthly intravitrealinjections.

The invention illustratively disclosed herein suitably may be practicedin the absence of any element, part, step, component, or ingredientwhich is not specifically disclosed herein.

While in the foregoing detailed description this invention has beendescribed in relation to certain preferred embodiments thereof, and manydetails have been set forth for purposes of illustration, it will beapparent to those skilled in the art that the invention is susceptibleto additional embodiments and that certain of the details describedherein can be varied considerably without departing from the basicprinciples of the invention.

What is claimed is:
 1. A method of delivering a compound to an eye, themethod comprising: applying to or into an eye of a mammal a compositionin a first physicochemical state, wherein the composition comprises atreatment agent microencapsulated in degradable microcapsules suspendedin a degradable thermo-responsive hydrogel, wherein the degradablemicrocapsules comprise polyethylene glycol (PEG), Mg(OH)₂, bovine serumalbumin, and sucrose; wherein the composition changes to a secondphysicochemical state upon administration, wherein the secondphysicochemical state is more solid than the first physicochemicalstate, and wherein the degradable microcapsules release themicroencapsulated treatment agent over time after applying.
 2. Themethod of claim 1, wherein the composition further comprises anon-encapsulated treatment agent dispersed within the degradablethermo-responsive hydrogel.
 3. The method of claim 1, wherein themicroencapsulated treatment agent comprises an anti-VEGF agent, ananti-PDGF agent, cells, delivery cells, antibiotic, a corticosteroid,enzymes, peptides, nucleic acids, or combinations thereof.
 4. The methodof claim 3, wherein the degradable microcapsules comprise copolymers ofpoly(lactic-co-glycolic acid) and chitin, polyethylene glycol, ordiacrylate.
 5. The method of claim 4, wherein the microencapsulatedtreatment agent comprises poly(lactic-co-glycolic acid), poly(lacticacid), polysaccharide chitin, alginate, or combinations or blockcopolymers thereof.
 6. The method of claim 1, wherein the hydrogelcomprises poly(N-isopropylacrylamide), poly(latic acid), polysaccharidechitin, alginate, diacrylate, or combinations or block copolymersthereof.
 7. The method of claim 1, wherein the second physicochemicalstate is degradable to release the microencapsulated treatment agent andfurther comprising controlling the degradation by type and/or amount ofcrosslinking to control the release of the microencapsulated treatmentagent.
 8. The method of claim 1, wherein the composition comprises asecond microencapsulated treatment agent suspended in thethermo-responsive hydrogel, the second microencapsulated treatment agenthaving a different rate of release than the microencapsulated treatmentagent.
 9. The method of claim 1, further comprising applying thecomposition in a first physicochemical state by intravitreal injection,by periocular or transcleral injection, by topical application, byintracameral application, by suprachoroidal application, within ocularimplants, or combinations thereof.
 10. The method of claim 1, furthercomprising applying the composition in a first physicochemical state bysmall gauge needle or microcatheter.
 11. The method of claim 1, furthercomprising applying the composition in a first physicochemical state asa scleral structure during retinal detachment treatment.
 12. The methodof claim 1, wherein the hydrogel is thermo-responsive at a physiologicaltemperature of about 32° C. to about 37° C.
 13. The method of claim 1,wherein the thermo-responsive hydrogels are in a liquid-like state atroom temperature and more solid state at body temperature.
 14. Themethod of claim 1, wherein the degradable microcapsules release themicroencapsulated treatment agent over 196 days after applying.
 15. Amethod of delivering a compound to an eye, the method comprising:applying to or into an eye of a mammal a composition in a firstphysicochemical state, wherein the composition comprises a treatmentagent microencapsulated in degradable microcapsules that are suspendedin a degradable thermo-responsive hydrogel, wherein thethermo-responsive hydrogels comprise a liquid-like state at roomtemperature and a more solid state at body temperature, and thesuspended degradable microcapsules comprise poly(lactic-co-glycolicacid), polyethylene glycol (PEG), Mg(OH)₂, bovine serum albumin, andsucrose; wherein the composition changes to a second physicochemicalstate upon administration, wherein the second physicochemical state ismore solid than the first physicochemical state, and wherein thedegradable microcapsules release the microencapsulated treatment agentover six months after applying.
 16. The method of claim 15, wherein thedegradable microcapsules comprise copolymers of thepoly(lactic-co-glycolic acid) and chitin, polyethylene glycol, ordiacrylate.
 17. The method of claim 15, wherein the secondphysicochemical state is degradable to release the microencapsulatedtreatment agent and further comprising controlling the hydrogeldegradation by type and/or amount of crosslinking to control the releaseof the microencapsulated treatment agent.
 18. The method of claim 17,wherein the composition comprises a second microencapsulated treatmentagent suspended in the thermo-responsive hydrogel, the secondmicroencapsulated treatment agent having a different rate of releasethan the microencapsulated treatment agent.
 19. The method of claim 17,wherein the composition further comprises a non-encapsulated treatmentagent dispersed within the degradable thermo-responsive hydrogel.
 20. Amethod of delivering a compound to an eye, the method comprising:applying to or into an eye of a mammal a composition in a firstphysicochemical state, wherein the composition includes amicroencapsulated treatment agent suspended in a degradablethermo-responsive hydrogel, wherein the microencapsulated treatmentagent comprises degradable microcapsules formed ofpoly(lactic-co-glycolic acid), polyethylene glycol (PEG), Mg(OH)₂,bovine serum albumin, and sucrose; wherein the composition changes to asecond physicochemical state upon administration, wherein the secondphysicochemical state is more solid than the first physicochemicalstate, and wherein the degradable microcapsules release themicroencapsulated treatment agent over time after applying.